Localised 1H/31P-MRS in Human Skeletal Muscle

All techniques applied to measure dynamic metabolic events involved in muscular contraction and recovery are indirect methods. Methods so far established include measurements of serum metabolites and gas exchange analysis, representing averaged metabolic changes for the whole body. More direct and localized methods are muscle biopsy, which can be used to sample the intracellular environment, and microdialysis or open-flow microperfusion, which samples muscle interstitial fluid. However, both techniques show significant limitations for experimental and clinical purposes, in particular their invasiveness. A noninvasive way of studying intracellular metabolism is Magnetic Resonance Spectroscopy (MRS). By using strong magnetic fields and various characteristic transmit/receive frequencies, biomolecules containing specific nuclei (e.g. 1H, 13C, 31P) may be studied in their natural environment in vivo. Over the last two decades several research facilities have developed large scale research programs to study metabolic events in the human muscle using 31 P-MRS. More recently, the additional techniques of 13C-MRS and 1H-MRS have been used to study various aspects of lactate, glucose and lipid metabolism in the human in vivo. The main limitations of 31 P-MRS are the inherently low sensitivity and the fact that lactate concentrations must be calculated indirectly using several model assumptions not yet fully validated in vivo. On the other hand, 1 H-MRS allows direct measurement of lactate, however, other intricacies are introduced. Although early attempts to combine the informations available from MRS of various nuclei (i.e., 1H, 19F, 23Na, 31 P) exist, it turned out that too much may be less, i.e., coil optimization is less effective if too many frequencies are used and uniform localisation is hampered by chemical shift differences. Improved MRS techniques optimized for a particular scientific question in combination with higher field strength, i.e. higher sensitivity and better spectral resolution, should allow to overcome or reduce several complications mentioned above and presented in more detail lateron. In this particular project we will focus an a combination of localised in vivo 1H and 31P MRS in human skeletal muscle under aerobic and anaerobic exercise which, together with the improved data quality, data quantity and models, should enhance our quantitative understanding of energy metabolism in human muscle.

When magnetic resonance spectroscopy (MRS) is applied to human tissue, metabolism can be studied in normal physiological and pathological states in vivo. MRS using the phosporus ( 31P) nucleus for excitation and signal detection as a noninvasive method has long been used to study cellular energy metabolism in vivo, paradigmatically in skeletal muscle. The major disadvantage of 31P MRS compared to needle biopsy has been the inability to quantify lactate directly. This has necessitated indirect 31P MRS approaches to important (and still much debated) issues in the control of glycolysis and cellular acid handling in vivo. An interleaved 31P- and 1 H MRS method was implemented by which lactate accumulation and the accompanying changes in phosphorus metabolites can be monitored simultaneously in a well defined gradient localised volume of interest positioned in a muscle during ischaemic (i.e. anaerobic) exercise and recovery on a custom built non- magnetic ergometer. Also interleaved is a standard localising 1 H pulse sequence, to monitor extra- and intramyocellular lipids, TMA and total creatine. In contrast to the vast majority of MRS studies investigating metabolism, where spectra of a single nucleus (commonly 1 H, 31P or 13C) were acquired or several MR spectra with different nuclei were measured in separate experiments, this work opens an additional "window" on muscle metabolism by interleaved localised acquisition of 1 H and 31P NMR spectra in a single experiment: Using this technique, the time courses of the concentrations of phosphocreatine, inorganic phosphate (Pi), ATP, total creatine, and lactate in human gastrocnemius muscle can be quantified directly, without using physiological model assumptions, while intracellular pH can be derived from the chemical shifts of Pi and PCr in 31P MR spectra. Quantifying intramuscular lactate by NMR spectroscopy is challenging, because the lactate signal is overlapped by strong lipid resonances which requires techniques like double quantum filter techniques and exhibits modulations which dependent on muscle fibre orientation relative to the magnetic field, intra- and extracellular compartmentation and relaxation times. Questions on metabolic efflux can be addressed when studying lactate, pH and PCr changes during recovery from ischaemic exercise with high time resolution, as well as control of glycogenolysis and coordination of oxidative versus glycolytic ATP production in aerobic and ischaemic exercise. In conclusion, the novel method gives an excellent fit both to biochemically measured lactate concentration in porcine gastrocnemius and to the lactate concentration in human calf muscle expected on the basis of changes in pH, Pi and PCr, helping to confirm model assumptions about cellular proton buffering capacity. Due to the localised measurement of 31P metabolites the acquired data are more specific for the exercised muscle compared to surface coil experiments without further localisation of the volume of interest. Finally, the larger amount of complementary information obtained from this triple interleaved experiment may be useful to study more subtle differences or specific changes in various physiological and pathological conditions. Perspectives are further improvement of specificity by multi nuclear spectroscopic imaging and an increase of sensitivity and time resolution by transferring the technique to a even higher field strength, and applications in sports medicine, rehabilitation and diabetes.